Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody-Drug Conjugate.

The utilization of antibody-drug conjugates (ADCs) has gained considerable attention in the field of targeted cancer therapy due to their ability to synergistically combine the specificity of monoclonal antibodies (mAbs) and the potency of small molecular drugs. However, the immunogenic nature of the antibody component within ADCs warrants the need for robust immunogenicity testing, including a neutralizing antibody (NAb) assay. Since the mechanism of action (MOA) of the ADC is to first bind to the target cells and then release the payload intracellularly to kill the cells, the most relevant NAb assay format would be a cell-based killing assay. However, in this paper, we present a case where a cell-based killing assay could not be developed after multiple cell lines and NAb-positive controls (PC) had been tested. Surprisingly, contrary to our expectations, all NAb PCs tested exhibited an increased killing effect on the target cells, instead of the expected protective response. This unexpected phenomenon most likely is due to the non-specific internalization of drug/NAb complexes via FcγRs, as an excessive amount of human IgG1 and mouse IgG2a, but not mouse IgG1, greatly inhibited drug or drug/NAb complexes induced cell death. To overcome this obstacle, we implemented a novel cell-based binding assay utilizing the Meso Scale Discovery (MSD) platform. We also propose that an in vitro cell killing NAb assay is limited to at best monitoring the target binding and internalization induced cell death, but not by-stander killing induced by prematurely released or dead-cell released payload, hence cannot really mimic the in vivo MOA of ADC.

A Novel Neutralization Antibody Assay Method to Overcome Drug Interference with Better Compatibility with Acid-Sensitive Neutralizing Antibodies.

Immunogenicity testing to detect and characterize anti-drug antibody (ADA) is required for almost all biotherapeutics. Monoclonal antibody biotherapeutics usually have long half-lives and for high-dose indications such as oncology, high level of drug will be present in the testing samples and interfere with ADA and/or neutralization antibody (NAb) measurement. To overcome this drug interference, acid-dissociation-based sample pre-treatment such as Bead-Extraction and Acid Dissociation (BEAD) has been successfully applied. The main concern for these acid-dissociation-based methods, however, is that harsh acid treatment could denature positive control Abs as well as NAb species in testing samples. In addition, high amount of biotinylated drug is needed in order to have effective competition with high level of drug in the samples, which in turn requires expensive magnetic beads. And the whole process of magnetic beads handling is tedious if doing manually and often causes trouble during assay transfer. Here, we describe a novel method which we named as Precipitation, Acid Dissociation and Biotin-drug as Assay Drug (PABAD). This novel method will need only one step of acid dissociation, with much milder and shorter acid treatment to maximally preserve NAb activity. In addition, only a fraction of biotinylated-drug is needed and there is no need to use additional streptavidin (SA)-plate or SA-magnetic beads for extraction. Compared to a BEAD-based assay, PABAD demonstrates significantly improved recovery of acid-sensitive NAb positive controls (PCs) and similar recovery of acid-resistant NAb PCs.

One-Step Regio- and Stereoselective Electrochemical Synthesis of Orexin Receptor Antagonist Oxidative Metabolites.

Synthesis of drug metabolites, which often have complex structures, is an integral step in the evaluation of drug candidate metabolism, pharmacokinetic (PK) properties, and safety profiles. Frequently, such synthetic endeavors entail arduous, multiple-step de novo synthetic routes. Herein, we present the one-step Shono-type electrochemical synthesis of milligrams of chiral α-hydroxyl amide metabolites of two orexin receptor antagonists, MK-8133 and MK-6096, as revealed by a small-scale (pico- to nano-mole level) reaction screening using a lab-built online electrochemistry (EC)/mass spectrometry (MS) (EC/MS) platform. The electrochemical oxidation of MK-8133 and MK-6096 was conducted in aqueous media and found to produce the corresponding α-piperidinols with exclusive regio- and stereoselectivity, as confirmed by high-resolution nuclear magnetic resonance (NMR) characterization of products. Based on density functional theory (DFT) calculations, the exceptional regio- and stereoselectivity for this electrochemical oxidation are governed by more favorable energetics of the transition state, leading to the preferred secondary carbon radical α to the amide group and subsequent steric hindrance associated with the U-shaped conformation of the cation derived from the secondary α-carbon radical, respectively.

Prediction of frozen virus stability based on degradation mechanisms, real-time data and modeling.

Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.

Neutralization Activity of Anti-drug Antibodies Against a Biotherapeutic Can Be Predicted from a Comprehensive Pharmacokinetics, Pharmacodynamics, and Anti-drug Antibody Data Analysis.

Historically, a neutralization antibody (NAb) assay is considered critical in immunogenicity assessment of biologic therapeutics, even with low anti-drug antibody (ADA) positive rates. In 2019, FDA new guidelines issued on immunogenicity testing acknowledged the possibility of using "a highly sensitive PD marker or an appropriately designed PK assay or both that generate data that inform clinical activity" to replace a NAb assay. In the current manuscript, we present data for PK, PD, and ADA assays which collectively succeed to replace the standalone NAb assay. The data include a total LC/MS-based PK assay, a serum neutralization antibody (SNA) assay that essentially measures pharmacodynamically functional PK and can detect NAb activity in the presence of 1:1 ratio of drug, and a highly drug-tolerant ADA assay. In addition, a model-based meta-analysis (MBMA) demonstrated that the ability of SNA assay to detect NAb at 1:1 ratio of drug is sensitive enough to monitor clinically meaningful efficacy change, which is 50% reduction of SNA titer. Our strategy of preparing a holistic data package discussed here may provide a roadmap to the community for alternatives in assaying neutralizing activity of ADA.

Diminishing GSH-Adduct Formation of Tricyclic Diazepine-based Mutant IDH1 Inhibitors.

Mutant isocitrate dehydrogenase 1 (IDH1) has been identified as an attractive oncology target for which >70% of grade II and III gliomas and ∼10% of acute myeloid leukemia (AML) harbor somatic IDH1 mutations. These mutations confer a neomorphic gain of function, leading to the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). We identified and developed a potent, selective, and orally bioavailable brain-penetrant tricyclic diazepine scaffold that inhibits mutant IDH1. During the course of in vitro metabolism studies, GSH-adduct metabolites were observed. The hypothesis for GSH-adduct formation was driven by the electron-rich nature of the tricyclic core. Herein, we describe our efforts to reduce the electron-rich nature of the core. Ultimately, a strategy focused on core modifications to block metabolic hot spots coupled with substitution pattern changes (C8 N → C linked) led to the identification of new tricyclic analogues with minimal GSH-adduct formation across species while maintaining an overall balanced profile.

Highly sensitive LC-MS method for stereochemical quality control of a pharmaceutical drug substance intermediate.

Stereochemical quality control for pharmaceutical drug substance intermediates is a daunting task, especially considering the need to separate multiple stereoisomers simultaneously with low ppm level sensitivity. To address these challenges, we have successfully implemented chiral column screening, and developed an ultrasensitive liquid chromatography-mass spectrometry (LC-MS) method to separate four stereoisomers including the API intermediate, its enantiomer, and two other diastereomers. Parameters such as mobile phase additives, MS fragmentor, and column temperature were optimized to achieve the desired selectivity and sensitivity. The method enabled stereoisomer detection with high sensitivity (2 ppm LOD and 5 ppm LOQ), good linearity, and desired spike recovery, and it has been successfully applied for stereoisomer quantitation in multiple large-scale batches and demonstrated chiral quality control of the drug substance intermediate.

Direct Drug Analysis in Polymeric Implants Using Desorption Electrospray Ionization - Mass Spectrometry Imaging (DESI-MSI).

PURPOSE: Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) coupled with gas-phase ion mobility spectrometry was used to characterize the drug distribution in polymeric implants before and after exposure to accelerated in vitro release (IVR) media. DESI-MSI provides definitive chemical identification and localization of formulation components, including 2D chemical mapping of individual components with essentially no sample preparation. METHODS: Polymeric implants containing 40% (w/w) entecavir and poly(D,L-lactide) (PLA) were prepared and then exposed to either acidified PBS (pH 2.5) or MeOH:H2O (50:50, v/v) medias during a 7-day IVR test using continuous flow-through (CFT) cell dissolution. The amount of drug released from the polymer matrix during the 7-day IVR test was monitored by online-ultraviolet spectroscopy (UV) and HPLC-UV. After that period, intact implants and radial sections of implants were analyzed by DESI-MSI with ion mobility spectrometry. The active ingredient along with impurities and contaminants were used to generate chemical maps before and after exposure to the release medias. RESULTS: Bi-phasic release profiles were observed for implants during IVR release using both medias. During the second phase of release, implants exposed to PBS, pH 2.5, released the entecavir faster than the implants exposed to MeOH:H2O (50:50, v/v). Radial images of the polymer interior show that entecavir is localized along the central core of the implant after exposure to MeOH:H2O (50:50, v/v) and that the drug is more uniformly distributed throughout the implant after exposure to acidified PBS (pH 2.5). CONCLUSIONS: DESI-MSI coupled with ion mobility analysis produced chemical images of the drug distribution on the exterior and interior of cylindrical polymeric implants before and after exposure to various release medias. These results demonstrated the utility of this technique for rapid characterization of drug and impurity/degradant distribution within polymeric implants with direct implications for formulation development as well as analytical method development activities for various solid parenteral and oral dosage forms. These results are especially meaningful since samples were analyzed with essentially no preparative procedures.

Probing in Vitro Release Kinetics of Long-Acting Injectable Nanosuspensions via Flow-NMR Spectroscopy.

Novel treatment routes are emerging for an array of diseases and afflictions. Complex dosage forms, based on active pharmaceutical ingredients (APIs) with previously undesirable physicochemical characteristics, are becoming mainstream and actively pursued in various pipeline initiatives. To fundamentally understand how constituents in these dosage forms interact on a molecular level, analytical methods need to be developed that encompass selectivity and sensitivity requirements previously reserved for a myriad of in vitro techniques. The knowledge of precise chemical interactions between drugs and excipients in a dosage form can streamline formulation development and process screening capabilities through the identification of properties that influence rates and mechanisms of drug release in a cost-effective manner, relative to long-term in vivo studies. Through this work, a noncompendial in vitro release (IVR) method was developed that distinguished the presence of individual components in a complex crystalline nanosuspension environment. Doravirine was formulated as a series of long-acting injectable nanosuspensions with assorted excipients, using low- and high-energy wet media milling methods. IVR behavior of all formulation components were monitored using a robust continuous flow-through (CFT) dissolution setup (USP-4 apparatus) with on-line 1H NMR end-analysis (flow-NMR). Results from this investigation led to a better understanding of formulation parameter influences on nanosuspension stability, surface chemistry, and dissolution behavior. Flow-NMR can be applied to a broad range of dosage forms in which specific molecular interactions from the solution microenvironment require further insight to enhance product development capabilities.

Development of a highly efficient decontamination approach for ceftolozane in the pharmaceutical manufacturing environment.

The ß-lactam core is a key structure responsible for inducing both IgE-mediated acute-onset hypersensitivity and T-cell-mediated delayed-onset hypersensitivity with penicillins in humans. There is essentially no clinically significant immunologic cross-reactivity noted between the ß-lactam cores of penicillins and cephalosporins based on challenge studies in humans. The side-chains appear to be more important in inducing IgE-mediated acute-onset hypersensitivity and T-cell delayed-onset hypersensitivity with cephalosporins in humans. Despite these clinical findings, the U. S. Food and Drug Administration (FDA) still requires the level of ß-lactam-related antibiotic residues to be controlled at very low levels in manufacturing facilities. Ceftolozane is Merck & Co., Inc., Kenilworth, NJ, USA's (MSD's) 5th generation broad spectrum cephalosporin antibiotic against gram-negative bacteria. In searching for the optimal decontamination method of ceftolozane, most methods were found to be very slow in opening the ß-lactam ring in ceftolozane. Moreover, most of the previously reported decontamination methods applied analytical methods that only monitored the disappearance of the parent molecule as the endpoint of degradation. In this way, many of the ß-lactam-containing degradation products could be overlooked. In order to develop an efficient decontamination solution for ceftolozane, a sensitive ultra high performance liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry (UHPLC-HRMS/MS) method was first developed to ensure the detection of the ß-lactam ring in all degradation products. Through online UHPLC-UV-HRMS monitoring, 2.5 N KOH in 50% aqueous MeOH or 50% aqueous EtOH was identified as the best condition to fully degrade the ß-lactam ring in ceftolozane. This decontamination could be done within 15 min, even at 100 mg/mL concentration, and thus enable a quick turnaround time for equipment cleaning in the ß-lactam manufacturing facility. This method was also successfully applied to 12 other commercially available ß-lactam antibiotics.

Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.

RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products.

Microbial biotransformation - an important tool for the study of drug metabolism.

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials. Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes. A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies. Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

In-Use Photostability Practice and Regulatory Evaluation for Pharmaceutical Products in an Age of Light-Emitting Diode Light Sources.

This article describes how the increased use of energy-efficient solid-state light sources (e.g., light-emitting diode [LED]-based illumination) in hospitals, pharmacies, and at home can help alleviate concerns of photodegradation for pharmaceuticals. LED light sources, unlike fluorescent ones, do not have spurious spectral contributions <400 nm. Because photostability is primarily evaluated in the International Council of Harmonization Q1B tests with older fluorescent bulb standards (International Organization for Standardization 10977), the amount of photodegradation observed can over-predict what happens in reality, as products are increasingly being stored and used in environments fitted with LED bulbs. Because photodegradation is premised on light absorption by a compound of interest (or a photosensitizer), one can use the overlap between the spectral distribution of a light source and the absorption spectra of a given compound to estimate if photodegradation is a possibility. Based on the absorption spectra of a sample of 150 pharmaceutical compounds in development, only 15% would meet the required overlap to be a candidate to undergo direct photodegradation in the presence of LED lights, against a baseline of 55% of compounds that would, when considering regular fluorescent lights. Biological drug products such as peptides and monoclonal antibodies are also expected to benefit from the use of more efficient solid-state lighting.

Enrichment of Relevant Oxidative Degradation Products in Pharmaceuticals With Targeted Chemoselective Oxidation.

The ability to produce and isolate relatively pure amounts of relevant degradation products is key to several aspects of drug product development: (a) aid in the unambiguous structural identification of such degradation products, fulfilling regulatory requirements to develop safe formulations (International Conference on Harmonization Q3B and M7); (b) pursue as appropriate safety evaluations with such material, such as chronic toxicology or Ames testing; (c) for a specified degradation product in a late-stage regulatory filing, use pure and well-characterized material as the analytical standard. Producing such materials is often a resource- and time-intensive activity, either relying on the isolation of slowly formed degradation products from stressed drug product or by re-purposing the drug substance synthetic route. This problem is exacerbated if the material of interest is an oxidative degradation product, because typical oxidative stressing (H2O2 and radical initiators) tends to produce a myriad of irrelevant species beyond a certain stress threshold, greatly complicating attempts for isolating the relevant degradation product. In this article, we present reagents and methods that may allow the rapid and selective enrichment of active pharmaceutical ingredient with the desired oxidative degradation product, which can then be isolated and used for purposes described above.

Accelerated Forced Degradation of Pharmaceuticals in Levitated Microdroplet Reactors.

Forced degradation is a method of studying the stability of pharmaceuticals in order to design stable formulations and predict drug product shelf life. Traditional methods of reaction and analysis usually take multiple days, and include LC-UV and LC-MS product analysis. In this study, the reaction/analysis sequence was accelerated to be completed within minutes using Leidenfrost droplets as reactors (acceleration factor: 23-188) and nanoelectrospray ionization MS analysis. The Leidenfrost droplets underwent the same reactions as seen in traditional bulk solution experiments for three chemical degradations studied. This combined method of accelerated reaction and analysis has the potential to be extended to forced degradation of other pharmaceuticals and to drug formulations. Control of reaction rate and yield is achieved by manipulating droplet size, levitation time and whether or not make-up solvent is added. Evidence is provided that interfacial effects contribute to rate acceleration.

Using miniature MS system with automatic blood sampler for preclinical pharmacokinetics study.

AIM: Preclinical pharmacokinetic studies are an essential part of modern drug development. In this work, we explored a new solution with onsite analysis using a miniature MS system, which can significantly improve the efficiency of the preclinical study. Materials & methods: A miniature mass spectrometer was used with an automatic blood sampler for onsite quantitation of drug compounds in whole blood samples. Slug-flow microextraction was used to replace the in-lab sample preparation. RESULTS & CONCLUSION: Animal studies were carried out using two drug compounds, using the auto sampler to take blood samples at preprogrammed time points. The miniature MS system was used to obtain drug concentrations, which were subsequently used to calculate the pharmacokinetic parameters.

Mixed Reality Meets Pharmaceutical Development.

As science evolves, the need for more efficient and innovative knowledge transfer capabilities becomes evident. Advances in drug discovery and delivery sciences have directly impacted the pharmaceutical industry, though the added complexities have not shortened the development process. These added complexities also make it difficult for scientists to rapidly and effectively transfer knowledge to offset the lengthened drug development timelines. While webcams, camera phones, and iPads have been explored as potential new methods of real-time information sharing, the non-"hands-free" nature and lack of viewer and observer point-of-view render them unsuitable for the R&D laboratory or manufacturing setting. As an alternative solution, the Microsoft HoloLens mixed-reality headset was evaluated as a more efficient, hands-free method of knowledge transfer and information sharing. After completing a traditional method transfer between 3 R&D sites (Rahway, NJ; West Point, PA and Schnachen, Switzerland), a retrospective analysis of efficiency gain was performed through the comparison of a mock method transfer between NJ and PA sites using the HoloLens. The results demonstrated a minimum 10-fold gain in efficiency, weighing in from a savings in time, cost, and the ability to have real-time data analysis and discussion. In addition, other use cases were evaluated involving vendor and contract research/manufacturing organizations.

Unusual (+/-)-electrospray ionization induced fragmentation: Structural elucidation of an in-process synthetic intermediate of doravirine (MK-1439) using liquid chromatography/high-resolution tandem mass spectrometry and two-dimensional nuclear magnetic resonance.

RATIONALE: During the development of a novel synthetic route to doravirine (1), a human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI), an unanticipated reaction intermediate, methyl (Z)-2-(3-chloro-5-cyanophenoxy)-5-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)pyridin-1(2H)-yl)-5-ethoxy-3-(trifluoromethyl)pent-2-enoate (2), was isolated. Moreover, an unusual electrospray ionization (ESI)-induced fragmentation was observed for 2. Hence, efforts were made towards the understanding of the structure of 2, which was crucial for the understanding of the reaction mechanism. METHODS: The isolated impurity was fully characterized by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC/HRMS/MS), hydrogen/deuterium (H/D) exchange, and an ensemble of two-dimensional nuclear magnetic resonance (2D-NMR) techniques. Density functional theory (DFT) calculations were also conducted. RESULTS: An unusual ESI-induced fragmentation was observed for intermediate 2, giving an ion for half of the molecule in the positive ion mode, with the other half of the molecule affording an ion in the negative ion mode. CONCLUSIONS: To the best of our knowledge, this unique ESI-induced fragmentation has not been previously reported in the literature. The underlying mechanism was explored and is supported by DFT calculations, which could greatly help the structural characterization of unknown impurities with similar structural features using ESI-MS in the future. Copyright © 2017 John Wiley & Sons, Ltd.

Mechanistic Study of the Gas-Phase In-Source Hofmann Elimination of Doubly Quaternized Cinchona-Alkaloid Based Phase-Transfer Catalysts by (+)-Electrospray Ionization/Tandem Mass Spectrometry.

An unusual in-source fragmentation pattern observed for 14 doubly quaternized cinchona alkaloid-based phase-transfer catalysts (PTC) was studied using (+)-ESI high resolution mass spectrometry. Loss of the substituted benzyl cation (R1 or R2) was found to be the major product ion [M2+ - R1+ or R2+]+ in MS spectra of all PTC compounds. A Hofmann elimination product ion [M - H]+ was also observed. Only a small amount of the doubly charged M2+ ions were observed in the MS spectra, likely due to strong Columbic repulsion between the two quaternary ammonium cations in the gas phase. The positive voltage in the MS inlet but not the ESI probe was found to induce this extensive fragmentation for all PTC diboromo-salts. Compound 1 was used as an example to illustrate the proposed in-source fragmentation mechanism. The mechanism of formation of the Hofmann elimination product ion [M - H]+ was further investigated using HRMS/MS, H/D exchange, and DFT calculations. The proposed formation of 2b as the major Hofmann elimination product ion was supported both by HRMS/MS and DFT calculations. Formation of product ion 2b through a concerted unimolecular Ei elimination pathway is proposed rather than a bimolecular E2 elimination pathway for common solution Hofmann eliminations. Graphical Abstract ᅟ.

Characterization of impurities of HIV NNRTI Doravirine by UHPLC-high resolution MS and tandem MS analysis.

World Health Organization estimates that 34 million individuals globally are living with Human Immunodeficiency Virus (HIV). Doravirine is a non-nucleoside reverse transcriptase inhibitors (NNRTI) being evaluated by Merck for the treatment of HIV-1 infection. Drug regulation authorities require the purity of a pharmaceutical to be fully defined. This is important to ensure that the pharmacological and toxicological effects are truly those of the drug substances and not because of the impurities. Thus, understanding the drug impurity profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. The impurity characterization can also provide useful information for critical assessment of pharmaceutical processes. Advances in mass spectrometry instrumentation and methods allow the identification of impurities in pharmaceuticals with a minimum of sample material and increased sensitivity. In this study, a rapid and sensitive method was developed for the structural determination of the major impurities of doravirine. The study utilizes ultra performance liquid chromatography-high-resolution-tandem mass spectrometry (UHPLC-HRMS/MS) techniques to perform structure elucidation of the unknown structures. This approach has significant impact on impurity structural elucidation, and a total of five trace-level impurities of doravirine were characterized using the developed method. Copyright © 2016 John Wiley & Sons, Ltd.